Increases in Ca2+ resulting from activation of intracellular Ca2+ channels regulate many physiological events. Dysfunction of these Ca2+ channels is involved in neurodegenerative and neurological disease, as well as exocrine, cardiac abnormalities and cancer. To appreciate how cell functions are controlled by Ca2+ signals, and how pathological cues subvert their function, we must understand how both the distribution and the properties (the 'functional architecture') of intracellular Ca2+ channels controls the patterning of Ca2+ signals. Here, we investigate the structural and functional coupling between two intracellular Ca2+-permeable channels that are activated by distinct second messengers: (i) nicotinic acid adenine dinucleotide phosphate (NAADP) which activates the recently discovered two-pore channels (TPCs) within endolysosomes, and (ii) inositol trisphosphate (IP3), which activates IP3 receptors (IP3Rs) in the endoplasmic reticulum. Despite localization in separate organelles, the activity of these Ca2+ channels is intimately related: a functional coupling garnering increasing attention in neurodegenerative disorders involving lysosomal proliferation. Here, by defining the mammalian TPC interactome we provide insight into two key unknowns: (i) how the functional architecture between TPCs and IP3Rs is established and (ii) the molecular identity of the NAADP receptor (NAADP-R, part of the TPC complex). Both are key pieces of knowledge for designing new drugs to modify this coupling. Our six person team, combining chemical, proteomic, molecular and live cell imaging expertise, will resolve: (1) Whether TPCs are Rab effectors? TPCs associate with a clade of Rab GTPases. We will define how TPC channels act as a node for coupling Ca2+ signaling to endolysosomal trafficking and fusion events. (2) How TPC/IP3R activity is coordinated between discrete organelles. We will use novel molecular insight from the TPC interactome to interrogate the functional architecture of TPCs/IP3Rs at membrane contact sites, and uncover how dysregulation triggers lysosomal proliferation. (3) Identify the NAADP-R. We have designed and optimized a novel bifunctional photoprobe to unmask the NAADP-R within the TPC interactome. This is a key roadblock, hampering knowledge of TPC activation. The broad significance of this work is in understanding principles controlling ion channel dynamics, and thereby the kinetics of Ca2+ signals that control compartmentalized cellular and system-levels outcomes. Such data will aid our understanding of the role of ubiquitous Ca2+ signaling pathways in health and disease.